The present invention is directed to the field of organic chemistry in general and specifically to the preparation of hydrophobic derivatives of cyclic ADP ribose.
Intracellular calcium plays key roles in stimulation-secretion coupling in pancreatic islet xcex2-cells. The elevation of cellular cytosolic calcium concentration ([Ca2+]c) is mediated through two pathways: Ca2+ release from intracellular calcium stores and Ca2+ influx from extracellular medium. The mechanisms underlying internal calcium release in xcex2-cells remain incompletely understood, and the relative contribution of intracellular Ca2+ release to the overall [Ca2+]c increase and subsequent insulin secretion needs to be determined.
Ca2+ release from intracellular stores is an important signaling mechanism for a variety of cellular processes and is generally controlled by two systems, the IP3 and cADPR systems (FIG. 1). IP3 acts directly on the IP3 receptor (IP3R) localized in the endoplasmic reticulum (ER). IP3R forms the Ca2+ releasing channel and regulates the efflux of Ca2+ from the ER to the cytosol. Cyclic ADP ribose increases the opening probability of other intracellular Ca2+ releasing channel formed by the ryanodine receptor (RyR) in the ER.
Ca2+ influx through voltage gated Ca2+ channels is a well-characterized phenomenon in xcex2-cells, and it is thought to play an important role in maintaining Ca2+ homeostasis, especially during glucose stimulation. However, contributions from internal calcium release cannot be ignored. Ca2+ influx from extracellular sources and Ca2+ release from the intracellular pool in human xcex2-cells has been examined, and showed that 42-75% of the increase in intracellular Ca2+ by glucose stimulation was due to the release of Ca2+ from the intracellular stores. Both IP3 and cADPR signaling systems have been reported in insulin secreting xcex2-cells, but controversies remain regarding which system is more important for maintaining proper insulin secretion responses.
To examine IP3 or cADPR induced Ca2+ release in xcex2-cells, it is necessary to deliver these second messengers inside cells and assay their effects on cellular calcium homeostasis and insulin secretion. Methods relying on triggering cell surface receptors to produce endogenous IP3 or cADPR inevitably activating other signaling pathways, making it impossible to separate the effects caused by IP3 or cADPR from those caused by other signaling branches. To deliver exogenous IP3 or cADPR inside cells, one need to overcome the difficulty of getting them across cell membranes. Both IP3 and cADPR are charged and hydrophilic molecules at physiological pH, thus are membrane impermeant. Previous techniques of getting these two molecules across hydrophobic cell membranes include microinjection, patch clamping, electroporation or detergent assisted permeabilization. All these methods are invasive and suffer from major drawbacks such as disrupting intact cell membranes, letting cytosolic factors leak out of cells, and compromising long term viability of cells. In addition, techniques such as microinjection or patch clamping can only be applied to single cells, making it practically impossible to study more physiological preparations such as islets.
One form of the present invention is a hydrophobic compound of the general formula: 
where R1, R2, R3 and R4 are each independently hydrogen or linear or branched alkyl groups having from 1 to 12 carbon atoms. R5 and R6 are each an alkyl group, metallic cation, a photo-labile caging group, or an acyloxymethylgroup or a homologue thereof. W is CH2, CF2, or CHF. X is N or CH. Y is N or CH. Z is chosen from the group including H, Br, NH2, OCH3, CH3 and N3.
Another form of the invention is a method for preparing a hydrophobic composition comprising the following steps: 
where RO and Rxe2x80x2O comprise independently in each location carboxylate groups further comprising from 2 to 20 carbon atoms.